Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
This invention relates to methods of diagnosing disorders characterized by known nucleic acid mutations. The instant methods employ the use of catalytic nucleic acid molecules, and are useful in connection with diagnosing such disorders as cancer and AIDS.
A variety of inherited and acquired diseases are associated with genetic variations such as point mutations, deletions and insertions. Some of these variations are directly associated with the presence of disease, while others correlate with disease risk and/or prognosis. There are more than 500 human genetic diseases which result from mutations in single genes (21, 22). These include cystic fibrosis, muscular dystrophy, xcex11-antitrypsin deficiency, phenylketonuria, sickle cell anemia or trait, and various other hemoglobinopathies (21, 22). Furthermore, individuals with increased susceptibility to several common polygenic conditions, such as atherosclerotic heart disease, have been shown to have an association with the inheritance of particular DNA sequence polymorphisms.
Cancer is thought to develop due to the accumulation of genetic lesions in genes involved in cellular proliferation or differentiation. The ras proto-oncogenes, K-ras, N-ras and H-ras, and the p53 tumor suppressor gene are examples of genes which are frequently mutated in human cancers. Specific mutations in these genes leads to an increase in transforming potential. Genetic analysis would be invaluable in the clinic for assessing disease risk, diagnosis of disease, predicting a patient""s prognosis or response to therapy, and monitoring a patient""s progress. The introduction of such genetic tests, however, will depend on the development of simple, inexpensive, and rapid assays for genetic variations.
Methods of in vitro nucleic acid amplification have wide-spread applications in genetics and disease diagnosis. In the last decade many techniques for amplification of nucleic acid have been described. These include the polymerase chain reaction (PCR) (1-7), the ligase chain reaction (LCR) (8), the strand displacement amplification assay (SDA) (9) and transcription-mediated amplification (TMA) (10, 11) (also known as self-sustained sequence replication (SSR)). The amplification products (amplicons) produced by PCR, LCR and SDA are DNA, whereas RNA amplicons are produced by TMA. DNA or RNA templates, generated by these protocols or others, can be analyzed for the presence of sequence variation (i.e. mutation) associated with the disease to be ascertained.
As with nucleic acid amplification, catalytic nucleic acids have been studied intensively in recent years. The potential for suppression of gene function using catalytic nucleic acids as therapeutic agents is widely discussed in the literature (12-18). Catalytic RNA molecules (ribozymes) have been shown to be capable of cleaving both RNA (12) and DNA (17) molecules. Similarly, catalytic DNA molecules (DNAzymes) have also been shown to be capable of cleaving both RNA (13, 19) and DNA (18) molecules. Catalytic nucleic acid can only cleave a target nucleic acid sequence, provided that target sequence meets minimum sequence requirements. The target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid and the target must contain a specific sequence at the site of cleavage. Examples of such sequence requirements at the cleavage site include the requirement for a purine:pyrmidine sequence for a class of DNAzyme cleavage (10-23 model) (19), and the requirement for the sequence uridine:H where H can equal A, C or U but not G, for the hammerhead ribozymes (23).
In addition to their therapeutic potential, catalytic nucleic acid molecules can also distinguish between targets which differ by a single point mutation (14-16). This is achieved by targeting a specific sequence which is present in wild-type but not mutant templates or vice versa. So far, this capacity for discrimination has only been exploited as a method for therapeutic manipulation of gene expression.
A review by Nollau-Wagener (24) compared several methodologies for the detection of point mutations with respect to the type of nucleic acid analyzed, the percentage of mutations detected, the time and cost of performing the assay, and problems relating to the use of toxic reagents. Each of the methodologies examined had its drawbacks. For example, denaturing gradient gel electrophoresis is time consuming, RNAase A cleavage can only detect about 70% of possible mutations, and chemical cleavage involves the use of toxic substances.
Another method, known as restriction fragment length polymorphism (RFLP), involves ascertaining whether a restriction enzyme site is present or absent at the locus of interest. In rare instances, mutations can be detected because they happen to lie within a naturally occurring restriction endonuclease recognition/cleavage site (31).
The inclusion of mismatched bases within primers used to facilitate in vitro amplification can result in the induction of artificial restriction endonuclease recognition/cleavage sites, and hence an increase in the number of loci which can be analyzed by RFLP (32). Modified primers containing mismatched bases have been used to induce artificial recognition/cleavage sites for restriction endonucleases at critical codons within the ras gene family (33-35). The general rules for designing primers which contain mismatched bases located near the 3xe2x80x2 termini of primers have been established (36).
Although the use of mismatched primers has expanded the utility of RFLP analysis, the technique is still limited by the fact that a minimum of four base pairs is required for recognition and cleavage by a restriction enzyme.
This invention provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a known nucleic acid mutation, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b)(i) amplifying the nucleic acid segment present in the isolated sample, which segment is known to contain the mutation in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segment with a catalytic nucleic acid molecule which specifically recognizes and cleaves a target sequence present either (1) in the nucleic acid segment having the known mutation or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether the catalytic nucleic acid molecule in step (b)(ii) cleaves the amplified segment, so as to determine whether the subject is afflicted with the disorder.
This invention also provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a plurality of known nucleic acid mutations, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b)(i) amplifying the nucleic acid segment present in the isolated sample, which segment is known to contain the plurality of mutations in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segment with a plurality of catalytic nucleic acid molecules, each of which specifically recognizes and cleaves a target sequence present either (1) in the nucleic acid segment having the known mutation or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether each of the catalytic nucleic acid molecules in step (b)(ii) cleaves the amplified segment, so as to determine whether the subject is afflicted with the disorder.
This invention further provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a plurality of known nucleic acid mutations, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b) (i) amplifying the nucleic acid segments present in the isolated sample, which segments collectively are known to contain the plurality of mutations in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segments with a plurality of catalytic nucleic acid molecules, each of which specifically recognizes and cleaves a target sequence present either (1) in one of the nucleic acid segments having one of the known mutations or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether each of the catalytic nucleic acid molecules in step (b)(ii) cleaves the amplified segment containing its respective target sequence, so as to determine whether the subject is afflicted with the disorder.
Finally, this invention provides kits for use in practicing the instant diagnostic methods. The first instant kit comprises (a) a catalytic nucleic acid molecule which specifically recognizes and cleaves a target sequence present either (i) in a nucleic acid segment having a mutation known to be characteristic of a disorder or (ii) in the corresponding wild-type nucleic acid segment, but not both, and (b) a nucleic acid reagent suitable for use in amplifying the nucleic acid segment containing the target sequence.
The second instant kit comprises (a) a 10-23 DNAzyme which specifically recognizes and cleaves a target sequence present either (i) in a nucleic acid segment having a mutation known to be characteristic of a disorder or (ii) in the corresponding wild-type nucleic acid segment, but not both, and (b) a DNA primer suitable for initiating amplification of the segment under polymerase chain reaction conditions, which primer contains at least one purine ribonucleotide residue which serves as the 5xe2x80x2 side of the site within the amplified segment recognized and cleaved by the 10-23 DNAzyme.
The third instant kit comprises (a) a first DNA primer which comprises a zymogene encoding a 10-23 DNAzyme that specifically recognizes and cleaves a target sequence present either (i) in a nucleic acid segment having a mutation known to be characteristic of a disorder or (ii) in the corresponding wild-type nucleic acid segment, but not both, which first primer is suitable for initiating amplification of the segment under polymerase chain reaction conditions; and (b) a second DNA primer suitable for initiating amplification of the segment under polymerase chain reaction conditions, which second primer contains at least one purine ribonucleotide residue which serves as the 5xe2x80x2 side of the site within the amplified segment recognized and cleaved by the 10-23 DNAzyme, such that, upon amplification, (i) the resulting amplified nucleic acid molecule comprises the 10-23 DNAzyme, and (ii) the amplified nucleic acid segment is recognized and cleaved in cis by the DNAzyme.
This invention provides methods employing catalytic nucleic acids to determine whether a subject is afflicted with a disorder characterized by the presence of one or more known nucleic acid mutations. These methods are collectively applicable to scenarios where the disorder is characterized by (i) a single mutation within a single nucleic acid segment, or (ii) a plurality of mutations within a single nucleic acid segment, or (iii) a plurality of mutations within a plurality of nucleic acid segments. For each mutation tested for by nucleic acid amplification, specific cleavage, and analysis, the instant methods provide a xe2x80x9cyes or noxe2x80x9d answer as to whether the mutation exists. This answer in turn ultimately leads to a xe2x80x9cyes or noxe2x80x9d answer as to whether the corresponding disorder is present in the subject.
Specifically, this invention provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a known nucleic acid mutation, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b)(i) amplifying the nucleic acid segment present in the isolated sample, which segment is known to contain the mutation in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segment with a catalytic nucleic acid molecule which specifically recognizes and cleaves a target sequence present either (1) in the nucleic acid segment having the known mutation or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether the catalytic nucleic acid molecule in step (b)(ii) cleaves the amplified segment, so as to determine whether the subject is afflicted with the disorder.
This invention also provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a plurality of known nucleic acid mutations, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b)(i) amplifying the nucleic acid segment present in the isolated sample, which segment is known to contain the plurality of mutations in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segment with a plurality of catalytic nucleic acid molecules, each of which specifically recognizes and cleaves a target sequence present either (1) in the nucleic acid segment having the known mutation or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether each of the catalytic nucleic acid molecules in step (b)(ii) cleaves the amplified segment, so as to determine whether the subject is afflicted with the disorder.
This invention further provides a method of determining whether a subject is afflicted with a disorder characterized by the presence of a plurality of known nucleic acid mutations, which comprises the steps of (a) isolating a sample of nucleic acid molecules from the subject; (b) (i) amplifying the nucleic acid segments present in the isolated sample, which segments collectively are known to contain the plurality of mutations in a subject afflicted with the disorder, and (ii) under suitable conditions, contacting the resulting amplified segments with a plurality of catalytic nucleic acid molecules, each of which specifically recognizes and cleaves a target sequence present either (1) in one of the nucleic acid segments having one of the known mutations or (2) in the corresponding wild-type nucleic acid segment, but not both, with the proviso that step (ii) can be performed either subsequent to or concurrently with step (i); and (c) determining whether each of the catalytic nucleic acid molecules in step (b)(ii) cleaves the amplified segment containing its respective target sequence, so as to determine whether the subject is afflicted with the disorder.
The instant methods can be used to diagnose disorders in any subject. As used herein, xe2x80x9csubjectxe2x80x9d means any animal, including, for example, mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In the preferred embodiment, the subject is a human.
The disorder diagnosed by the instant invention can be any disorder characterized by the presence of at least one known nucleic acid mutation, which mutation is missing when such disorder is absent. Such disorders are well known in the art and include, by way of example, cancer, AIDS, cystic fibrosis, muscular dystrophy, xcex11-anti-trypsin deficiency, phenylketonuria, sickle cell anemia or trait, and various other hemoglobinopathies. In one embodiment, the disorder is selected from the group consisting of cancer, AIDS, and cystic fibrosis. In the preferred embodiment, the disorder is cancer. In the Experimental Details section which follows, numerous examples are given of specific mutations, target sequences containing same, and catalytic nucleic acids used for diagnosing such disorders as cancer, AIDS and cystic fibrosis.
As used herein, xe2x80x9ccatalytic nucleic acid moleculexe2x80x9d means a DNA molecule (also known in the art as a xe2x80x9cDNAzymexe2x80x9d) or RNA molecule (also known in the art as a xe2x80x9cribozymexe2x80x9d) which specifically recognizes and cleaves a distinct target nucleic acid sequence. For both DNAzymes and ribozymes, the target nucleic acid sequence can be either DNA or RNA.
The nucleic acid sequence in which the known disorder-characterizing mutation(s) resides (i.e., the sequence amplified in the instant methods) can be a DNA or RNA sequence. These mutation(s) include, for example, point mutations, deletion mutations, insertion mutations and frame-shift mutations. Each of the amplified nucleic acid segment and catalytic nucleic acid molecule can be either DNA or RNA. In one embodiment, the amplified nucleic acid segment is RNA and the catalytic nucleic acid molecule is either DNA or RNA. In a further embodiment, the amplified nucleic acid segment is DNA and the catalytic nucleic acid molecule is either RNA or DNA (25).
Methods for isolating and amplifying nucleic acid molecules used in the instant invention are well known in the art. More specifically, methods of isolating a sample of nucleic acid molecules from the subject include, for example, phenol chloroform extraction, quick lysis, capture on columns and polymer capture (20, 26-29). Methods of amplifying a nucleic acid sequence include, for example, PCR, LCR, SDA and TMA (also known as (SSR)) (1-11).
Suitable conditions for contacting an amplified nucleic acid segment containing a target sequence with a catalytic nucleic acid molecule so as to permit specific recognition and cleavage of the target sequence are well known in the art. In addition, such conditions are exemplified in the Experimental Details section below.
Methods of determining whether a catalytic nucleic acid molecule cleaves an amplified nucleic acid segment are also routine in the art. Such methods include, by way of example, polyacrylamide gel electrophoresis and capillary electrophoresis (20, 30).
In the preferred embodiment of this invention, (a) the amplification is performed using a polymerase chain reaction; (b) the catalytic nucleic acid molecule is a 10-23 DNAzyme; and (c) the polymerase chain reaction employs a DNA primer (i.e., a xe2x80x9cchimericxe2x80x9d primer) suitable for initiating amplification of the segment, which primer contains at least one purine ribonucleotide residue which serves as the 5xe2x80x2 side of the site within the amplified segment recognized and cleaved by the 10-23 DNAzyme. This purine ribonucleotide residue in the chimeric primer is required for cleavage by the 10-23 DNAzyme. Thus, using this chimeric primer permits the 10-23 DNAzyme cleavage site to be generated in a PCR reaction. The chimeric primer can also include, for example, a ribonucleotide residue that serves as the 3xe2x80x2 side of the site recognized and cleaved by the 10-23 DNAzyme.
In one form of this embodiment, the amplified segment is recognized and cleaved in trans by the DNAzyme. In another form, (a) the polymerase chain reaction employs a second DNA primer suitable for initiating amplification of the segment, which second primer comprises a zymogene encoding a 10-23 DNAzyme such that, upon amplification, the resulting amplified nucleic acid molecule comprises the 10-23 DNAzyme; and (b) the amplified nucleic acid segment is recognized and cleaved in cis by the DNAzyme.
As used herein, xe2x80x9ccisxe2x80x9d cleavage by a DNAzyme shall mean that the DNAzyme recognizes and cleaves a sequence coexisting therewith on the same amplified nucleic acid molecule. Trans cleavage shall mean that the DNAzyme cleaves a substrate located on a different molecule. Finally, xe2x80x9czymogenexe2x80x9d shall mean a nucleic acid sequence which comprises the anti-sense (i.e. complementary) sequence of a catalytic nucleic acid molecule, and whose transcription product is the catalytic nucleic acid molecule itself.
This invention still further provides kits for use in practicing the instant diagnostic methods. The first instant kit comprises (a) a catalytic nucleic acid molecule which specifically recognizes and cleaves a target sequence present either (i) in a nucleic acid segment having a mutation known to be characteristic of a disorder or (ii) in the corresponding wild-type nucleic acid segment, but not both, and (b) a nucleic acid reagent suitable for use in amplifying the nucleic acid segment containing the target sequence.
In one embodiment, the kit comprises a plurality of catalytic nucleic acid molecules. The nucleic acid reagent suitable for use in amplifying the nucleic acid segment containing the target sequence can be, for example, a nucleic acid primer. In one embodiment, the kit comprises a plurality of such nucleic acid reagents.
More specifically, the second instant kit comprises (a) a 10-23 DNAzyme which specifically recognizes and cleaves a target sequence present either (i) in a nucleic acid segment having a mutation known to be characteristic of a disorder or (ii) in the corresponding wild-type nucleic acid segment, but not both, and (b) a DNA primer suitable for initiating amplification of the segment under polymerase chain reaction conditions, which primer contains at least one purine ribonucleotide residue which serves as the 5xe2x80x2 side of the site within the amplified segment recognized and cleaved by the 10-23 DNAzyme.
The third instant kit comprises (a) a first DNA primer which comprises a zymogene encoding a 10-23 DNAzyme that specifically recognizes and cleaves a target sequence present either (i) in a nucleic acid segment having a mutation known to be characteristic of a disorder or (ii) in the corresponding wild-type nucleic acid segment, but not both, which first primer is suitable for initiating amplification of the segment under polymerase chain reaction conditions; and (b) a second DNA primer suitable for initiating amplification of the segment under polymerase chain reaction conditions, which second primer contains at least one purine ribonucleotide residue which serves as the 5xe2x80x2 side of the site within the amplified segment recognized and cleaved by the 10-23 DNAzyme, such that, upon amplification, (i) the resulting amplified nucleic acid molecule comprises the 10-23 DNAzyme, and (ii) the amplified nucleic acid segment is recognized and cleaved in cis by the DNAzyme.
In one embodiment, the instant kits further comprise one or more of the following: (a) reagents useful for isolating a sample of nucleic acid molecules from a subject being diagnosed; (b) reagents useful for amplifying a nucleic acid segment present in the isolated sample, which segment is known to contain a mutation in a subject afflicted with the disorder; and (c) reagents useful for creating suitable reaction conditions for catalytic nucleic acid activity. The reagents in components (a)-(c) of the instant kits can either be obtained commercially or made according to well known methods in the art, as exemplified in the Experimental Details section below.
The components of the instant kits can be in solution or lyophilized as appropriate. In one embodiment, the components of the instant kits are in the same compartment, and in another embodiment, the components of the instant kit are in separate compartments. In the preferred embodiment, the kits further comprise instructions for use.